CN107652194B - A class of compounds for the treatment of neurological disorders - Google Patents

Abstract

The invention belongs to the technical field of medicine, and relates to a compound with a structure shown in a formula I and pharmaceutically acceptable salts thereof,

Description

A class of compounds for the treatment of neurological disorders

technical field

The invention belongs to the technical field of medicine, and more specifically relates to a class of compounds with neuron damage protection, a preparation method thereof, a pharmaceutical composition containing them, and their use as a medicine for nervous system diseases.

Background technique

"Cerebral stroke" (cerebral stroke), also known as "stroke", "cerebral vascular accident" (CVA), is an acute cerebrovascular disease caused by the sudden rupture of blood vessels in the brain or the inability of blood to flow to the brain due to vascular blockage. A group of disorders that damage brain tissue, including ischemic and hemorrhagic stroke. Stroke has become one of the main causes of death in my country, and ischemic stroke accounts for 60-80% of strokes. It has the characteristics of high disability and high fatality. Therefore, the treatment of stroke has become an important research topic for neurological diseases. In addition to acute thrombolysis, neuroprotective agents have important implications for the rehabilitation of stroke patients.

The drugs currently studied for neuroprotection mainly include glutamate receptor antagonists, free radical scavengers, calcium channel antagonists, gamma-aminobutyric acid (GABA) agonists, citicoline, inflammatory response inhibitors, Estrogen, erythropoietin (EPO), etc., some experiments have proved that they have a certain neuroprotective effect, and the clinical effect needs further research. There is an urgent need to develop new drugs as neuroprotective agents for the treatment of stroke.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to develop new compounds with neuroprotective effect for preparing medicines for the treatment of stroke and other nervous system diseases.

In order to solve the above-mentioned problems, the present invention provides a compound having the structure of formula I and a pharmaceutically acceptable salt thereof:

in:

R1 and R2 are: hydrogen, halogen;

R3 is: hydrogen, alkyl, benzyl, phenethyl.

The compounds of the present invention having the structure of formula I and their pharmaceutically acceptable salts, the preferred compounds of formula I are:

The compounds of the formula I of the present invention and their pharmaceutically acceptable salts, the pharmaceutically acceptable salts refer to: the compounds form salts with inorganic acids and organic acids.

In particular, the pharmaceutically acceptable salts of the present invention refer to: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, phosphate, acetate, propionate, butyrate, Lactate, mesylate, p-toluenesulfonate, maleate, benzoate, succinate, tartrate, citrate, fumarate, taurate, gluconate.

The preparation method of the compound of formula I of the present invention, wherein intermediate II is reacted at room temperature in the presence of liquid bromine and chloroform to generate intermediate III, intermediate III and intermediate IV are reacted at room temperature in the presence of acetonitrile and sodium bicarbonate, and quenched by adding water The reaction was quenched, extracted, washed, and purified to obtain compound I,

其中R1、R2、R3如权利要求1所述。

wherein R1, R2 and R3 are as described in claim 1.

Another preparation method of the compound of formula I of the present invention, wherein intermediate II is reacted in the presence of ethyl acetate and copper bromide to form intermediate III, intermediate III and intermediate IV are in N,N-dimethylformamide and In the presence of sodium bicarbonate, react at room temperature, add water to quench the reaction, extract, wash, and purify to obtain compound I,

wherein R1, R2 and R3 are as described in claim 1.

The present invention also relates to a pharmaceutical composition for treating nervous system diseases, which comprises a therapeutically effective amount of any one of the compounds having the structure of formula I or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers . Wherein the neurological disease is stroke.

In addition, the present invention also relates to the use of the compound having the structure of formula I and a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of nervous system diseases. Wherein the neurological disease is stroke.

Although the compounds of the present invention can be administered directly without any formulation, the various compounds described are preferably used in the form of pharmaceutical formulations, which may be administered parenterally (eg, intravenously, intramuscularly) and orally.

Pharmaceutical compositions of the compounds of the present invention are prepared by combining the compounds of the present invention with a pharmaceutically acceptable solid or liquid carrier, and optionally with pharmaceutically acceptable auxiliaries and excipients, using standard and conventional techniques The formulations are combined into microparticles or microspheres. Solid dosage forms include tablets, dispersible granules, capsules, sustained-release tablets, sustained-release pellets, and the like. A solid carrier can be at least one substance which may act as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, disintegrating agent, and encapsulating agent. Inert solid carriers include magnesium phosphate, magnesium stearate, sugar syrup, lactose, pectin, propylene glycol, polysorbate 80, dextrin, starch, gelatin, cellulosics such as methylcellulose, microcrystalline cellulose, Low melting point paraffin, polyethylene glycol, mannitol, cocoa butter, etc. Liquid dosage forms include solvents, suspensions such as injections, powders and the like.

The amount of the active ingredient (compound of the present invention) contained in the pharmaceutical composition and unit dosage form can be specifically applied according to the patient's condition and the situation diagnosed by the doctor, and the amount or concentration of the compound used can be adjusted within a wide range.

The compound having the structure of formula I or a pharmaceutically acceptable salt thereof of the present invention has obvious neuroprotective effect, and is of great significance to stroke and other nervous system diseases.

The neuroprotective effects of the compounds of the present invention are further described below through pharmacodynamic experiments.

1 Materials and methods:

1.1 Experimental materials

1.1.1 Experimental animals The rats used in the experiment were SPF grade Sprague Dawley rats, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Among them, gestation day 16-18 rats were used for neuronal culture.

1.1.2 Main reagents and instruments DMEM medium, Neurobasal medium, fetal bovine serum, and 0.25% trypsin were purchased from Invitrogen. Cell culture plates, cell culture flasks and centrifuge tubes were purchased from Corning Company. 2,2-Azobis(2-methylpropylimididine)dihydrochloride (2,2′-Azobis(2-methylpropionamidine)dihydrochloride, AAPH), CCK-8 cell viability kit was purchased from Dongren Company.

1.2 Experimental method

1.2.1 Primary Neuron Culture

The 16-18-day-old gestational rats were anesthetized with 10% chloral hydrate, placed in an ultra-clean workbench, and the embryos were isolated under aseptic conditions and transferred to Neuralbasal medium. Take the cortex, remove the meninges and blood vessels, wash twice with medium, cut into pieces, disperse the cells by pipetting, digest with 0.25% trypsin for 10 minutes, remove all tissue pieces by filtration, and suspend the cells in DMEM containing 10% fetal bovine serum. medium, seeded in 24-well culture plates, the medium in the plate was removed the next day, and replaced with neuronal medium: Neuralbasal medium containing 2% B-27 supplement, and the medium was changed every other day. used for experiments after 7 days.

1.2.2 AAPH-induced cell damage and cell viability detection:

2,2-Azobis(2-methylpropylimididine) dihydrochloride (2,2′-Azobis(2-methylpropionamidine) dihydrochloride, AAPH) in DMEM to prepare 1mM; 5mM; 10mM; 20mM and 40mM The solution was added to the neurons respectively, and CCK-8 solution was added at the same time. After 4 hours of action, the absorbance at 495nm wavelength was measured with a spectrophotometer to analyze cell viability and observe the effect of AAPH on cell viability.

1.2.3 The protective effect of the new compound on neurons.

Primary cultured neurons were first induced with 40 mM AAPH for 4 h. The medium was changed and new compounds D6, D14, D21, D22 were added at different concentrations, where each compound was at a low concentration of 1 μM, a medium concentration of 10 μM, and a high concentration of 100 μM. After 24 hours of protection, CCK-8 solution was added, and after 4 hours of action, the absorbance at a wavelength of 495 nm was measured with a spectrophotometer to analyze cell viability and determine the protective effect on neurons.

1.2.4 Anti-apoptotic effects of new compounds

Primary cultured neurons were first induced with 40 mM AAPH for 4 h. The medium was changed and new compounds D6, D14, D21, D22 were added at different concentrations, where each compound was at a low concentration of 1 μM, a medium concentration of 10 μM, and a high concentration of 100 μM. After 24 hours of protection, anti-apoptosis detection reagent solution was added, and then the number of apoptotic cells was observed under a fluorescence microscope, and normal cells and apoptotic cells were recorded in 10 fields of view, and their percentages were calculated. In order to determine the anti-apoptotic effect of the new compound.

1.2.5 Statistics and Analysis

SPSS15.0 was used for statistical analysis of the experimental results. The experimental data were expressed as mean±standard deviation, and the comparison between groups was analyzed by variance analysis. P<0.05 was statistically significant.

Table 1 Protective effect of the compounds of the present invention on AAPH-induced neurons

Table 2 Anti-apoptotic effect of the compounds of the present invention on AAPH-induced neurons

2. Results:

2.1 AAPH can simulate stroke to prepare neuronal injury model

Primary cultured neurons express neuron-specific marker neurofilament protein (Neurofilament, NF), indicating that the cultured cells are neurons, and different concentrations of AAPH can cause damage to neurons, and cell activity decreases with the increase of AAPH concentration . Among them, 40mM AAPH can obviously induce neuronal damage.

2.2 New compounds can reduce neuronal damage

After 40mM AAPH-induced neuronal injury, cerebroside carnosine-astroglial conditioned medium was added for 24h. Neuronal cell viability was then detected. The results showed that the new compounds (Table 1) were able to reverse the neuronal damage caused by AAPH, and the results were statistically significant.

2.3 New compounds can reduce apoptosis

Through the anti-apoptotic effect test of the new compound in 1.2.4, the percentage of apoptotic cells was counted (Table 2), indicating that the compound of the present invention has an anti-apoptotic effect on cells.

Detailed ways

Reference will now be made in detail to various embodiments of the present invention, and although the invention has been described in conjunction with exemplary embodiments, it should be understood that this specification is not intended to limit the invention to these exemplary embodiments. On the contrary, the invention is intended to cover not only the exemplary embodiments, but also various alternatives, modifications, equivalents and other embodiments, which are included within the spirit and scope of the invention as defined by the appended claims.

Example 1

Preparation of Compound D6 of the Invention

6.7g (40mmol) D1, 16.6g (120mmol) potassium carbonate, 30ml DMF were added to a 100ml pear-shaped bottle successively, and after stirring at 40 degrees Celsius for 10 minutes, 5.2ml (44mmol) D2 was added, and the reaction was performed at 40 degrees Celsius for 4h, cooled to room temperature, Ice water was added to the reaction flask, filtered, washed with water, and dried to obtain 12 g (39.1 mmol) of white powdery solid product D3, with a yield of 97.7%;

1H NMR (500MHz, CDCl3) δ 2.55 (s, 3H), 3.95 (s, 3H), 5.23 (s, 2H), 6.89 (d, J=8.0Hz, 1H), 7.32, (m, 1H), 7.38 (t, 1H), 7.43 (m, 2H), 7.50 (br.d., 1H), 7.55 (br.s., 1H);

Add 2048 (8mmol) D3 and 50ml of chloroform into a 200ml pear-shaped bottle in turn, dissolve 0.5ml (8mmol) of liquid bromine in 50ml of chloroform at room temperature and slowly add it dropwise to a reaction flask, and react for 2h after the addition is complete. After the end, it was washed with saturated sodium thiosulfate, washed with saturated sodium chloride solution, and purified with silica gel column to obtain 1.4 g of white powdery product D4 with a yield of 52.4%.

1H NMR (500MHz, DMSO-d6) δ 3.82(s, 3H), 4.85(s, 2H), 5.21(s, 2H), 7.18(d, J=8.0Hz, 1H), 7.40(m, 3H) , 7.45(m, 2H), 7.49(br.s., 1H), 7.66(br.d., J=8.0Hz, 1H);

334 mg (1 mmol) D4, 5 ml acetonitrile, 0.1 ml p-fluoroaniline (1.1 mmol), 168 mg sodium bicarbonate were successively added to a 50 ml pear-shaped bottle, and the reaction was carried out at room temperature for 12 h. The reaction was quenched by adding water, and extracted with ethyl acetate for 3 times. It was washed with sodium chloride solution and purified by silica gel column to obtain 180 mg of product D6 as light yellow powder with a yield of 51.9%.

D6 Chemical information: Molecular formula: C22H20FNO3, molecular weight: 365, purity: 98%, appearance: light yellow powder.

Spectral data: 1H NMR (500MHz, CDCl3)δ3.95(s,3H), 4.53(s,2H), 5.26(s,2H), 6.66(m,2H), 6.93(m,3H), 7.33(m ,1H),7.39(m,3H),7.44(m,3H),7.5 6(m,2H);13C NMR(500MHz, DMSO-d6)δ50.59,56.33,70.55,111.14,113.08,113.91,115.69 ,115.87,123.00, 128.54,128.70,128.84,129.16,137.16,145.55,149.57,152.96,154.23,156.06,1 95.09

Example 2

Preparation of compound D14 of the present invention

2656mg (16mmol) D1, 50ml ethyl acetate, 7.0g (28mmol) copper bromide were successively added into a 100ml eggplant-shaped flask, stirred and refluxed for 5h, the reactant was cooled and filtered with celite, and the filtrate was sequentially washed with saturated sodium thiosulfate The solution was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, evaporated to dryness under reduced pressure, and purified by silica gel column to obtain 2.2 g of white powdery product D11, with a yield of 56%;

370mg (1.5mmol) of D11, 5ml of N,N-dimethylformamide, 0.2ml of p-fluoroaniline (2.0mmol), and 252mg of sodium bicarbonate were successively added to a 50ml pear-shaped bottle, and the reaction was carried out at room temperature for 3h, and water was added to quench the reaction. It was extracted three times with ethyl acetate, washed with saturated sodium chloride solution, and purified with silica gel column to obtain 200 mg of product D14 as a yellow powder with a yield of 48.5%.

D14 Chemical information: Molecular formula: C15H14FNO3, molecular weight: 275, purity: 98%, appearance: light yellow powder.

Spectral data: 1H NMR (500MHz, DMSO-d6) δ3.86(s,3H), 4.56(s,2H), 5.73(s,1H), 6.68(m,2H), 6.92(m,3H), 7.54 (d, J=2.0Hz, 1H), 7.65 (dd, J=2.0, 8.0Hz, 1H), 10.03 (s, 1H); 13C NMR (500 MHz, DMSO-d6) δ 50.4356.37, 111.80, 113.89, 115.71, 115.86, 123.42, 127.55, 145.60, 148.25, 152.70, 154.14, 155.97, 195.28

Example 3

Preparation of compound D21 of the present invention

See Example 2 for the preparation of compound D11

Add 370mg (1.5mmol) of D11, 5ml of N,N-dimethylformamide, 0.2ml of 2,4-difluoroaniline (2.0mmol), and 252mg of sodium bicarbonate into a 50ml pear-shaped bottle in turn, react at room temperature for 3h, and add water. The reaction was quenched, extracted three times with ethyl acetate, washed with saturated sodium chloride solution, and purified by silica gel column to obtain 240 mg of product D21 as a yellow powder with a yield of 54.6%.

D21 Chemical information: Molecular formula: C15H13F2NO3, molecular weight: 293, purity: 98%, appearance: yellow powder.

Spectral data: 1H NMR (500MHz, DMSO-d6) δ3.84(s, 3H), 4.60(d, J=5.5Hz, 2H), 5.34(s, 1H), 6.70(m, 1H), 6.81(m , 1 H), 6.87(d, J=8.0Hz, 1H), 7.08(m, 1H), 7.50(br.s., 1H), 7.60(dd, J=2.0, 8.0Hz, 1H), 10.03( s, 1H); 13C NMR (500MHz, DMSO-d6) δ49.70, 56.17, 103.77, 111.15, 113.08, 115.54, 123.29, 127.1 2, 133.69, 148.08, 150.50, 152.64, 153.47, 194.71

Example 4

Preparation of compound D22 of the present invention

See Example 2 for the preparation of compound D11

370mg (1.5mmol) of D11, 5ml of N,N-dimethylformamide, 254mg (2mmol) of 4-chloroaniline, 252mg of sodium bicarbonate were successively added to a 50ml pear-shaped bottle, and the reaction was carried out at room temperature for 3h. Water was added to quench the reaction, and acetic acid was added. It was extracted three times with ethyl ester, washed with saturated sodium chloride solution, and purified by silica gel column to obtain 160 mg of the product D22 in the form of light yellow powder with a yield of 55.0%.

D22 Chemical information: Molecular formula: C15H14ClNO3, molecular weight: 291, purity: 98%, appearance: white powder.

Spectral data: 1H NMR (500MHz, DMSO-d6) δ3.84 (s, 3H), 4.57 (s, 2H), 6.68 (d, J=8.0Hz, 1H), 6.89 (d, J=8.0Hz, 1H) ), 7.08(d, J=8.0Hz, 1H), 7.52(br.s., 1H), 7.62(br.d., 1H), 10.03(s, 1H); 13C NMR(500MHz, DMSO-d6) δ49.57,55.99,111.43,114.15,115.34,119.53,123.06,127.1 0,128.71,147.49,147.86,152.35,194.61

Certain exemplary embodiments of the present invention have been presented above for purposes of illustration. The above description is not intended to be exhaustive of the invention, nor is it intended to limit the invention to the precise form disclosed. Obviously, many changes and variations are possible in light of the above description. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application, to thereby enable others skilled in the art to make and use various exemplary embodiments of the invention, as well as various alternatives and modifications. Instead, the scope of the invention is defined by the appended claims and their equivalents.

Claims (9)

1. Compounds having the structure of formula I and pharmaceutically acceptable salts thereof:

in: When R1 is fluorine, R2 is hydrogen, and R3 is benzyl, the following compounds are represented:

When R1 is fluorine, R2 is hydrogen, and R3 is hydrogen, the following compounds are represented: When R1 is fluorine, R2 is fluorine, and R3 is hydrogen, the following compounds are represented:

When R1 is chlorine, R2 is hydrogen, and R3 is hydrogen, the following compounds are represented: . 2. The compound having the structure of formula I according to claim 1 and a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt refers to: the compound forms a salt with an inorganic acid or an organic acid. 3. The compound according to claim 2 and a pharmaceutically acceptable salt thereof, said pharmaceutically acceptable salt refers to: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, Phosphate, acetate, propionate, butyrate, lactate, mesylate, p-toluenesulfonate, maleate, benzoate, succinate, tartrate, citrate , Fumarate, Taurine, Gluconate. 4. the preparation method of formula I compound in claim 1 is characterized in that: intermediate II reacts at room temperature in the presence of liquid bromine and chloroform to generate intermediate III, intermediate III and intermediate IV in the presence of acetonitrile and sodium bicarbonate , react at room temperature, add water to quench the reaction, extract, wash, and purify to obtain compound I,

wherein R1, R2 and R3 are as described in claim 1. 5. the preparation method of the compound of formula I in claim 1, it is characterized in that: intermediate II reacts to generate intermediate III in the presence of ethyl acetate and copper bromide, intermediate III and intermediate IV are in N,N-dimethyl In the presence of formamide and sodium bicarbonate, react at room temperature, add water to quench the reaction, extract, wash, and purify to obtain compound I, wherein R1, R2 and R3 are as described in claim 1. 6. A pharmaceutical composition for treating nervous system diseases, comprising a therapeutically effective amount of the compound having the structure of formula I as claimed in claim 1 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers. 7. The pharmaceutical composition of claim 6, wherein the neurological disease is stroke. 8. Use of the compound having the structure of formula I and pharmaceutically acceptable salts thereof according to claim 1 in the preparation of a medicament for the treatment of nervous system diseases. 9. The use according to claim 8, wherein the neurological disease is stroke.